Novel medicinal use of alpha antigen or alpha antigen gene

ABSTRACT

The α antigen-encoding gene and the α antigen protein suppress the production of interleukin-4 etc., improve the Th2 type cytokine-dominant state, and furthermore inhibit various conditions of allergic diseases such as IgE production, histamine release and eosinophil infiltration, and therefore they are very effective for the prevention or treatment of atopic diseases such as atopic dermatitis, asthma, allergic rhinitis, and allergic conjunctivitis, and more broadly allergic diseases.

TECHNICAL FIELD

[0001] The present invention relates to novel pharmaceutical uses of αantigen derived from acid-fast bacteria (Mycobacteria) or analogsthereof or genes encoding them. More specifically, it relates to novelpharmaceutical uses of α antigen derived from Mycobacterium kansasii oranalogs thereof or expression vectors containing a gene encoding themfor the prevention or treatment of allergic diseases such as atopicdermatitis, asthma, allergic rhinitis and allergic conjunctivitis.

BACKGROUND ART

[0002] Allergic diseases such as atopic dermatitis, asthma, allergicrhinitis and allergic conjunctivitis are diseases in whichhypersensitive reactions occur against environmental antigens to whichnormal healthy people do not react and destruction and disorders ofvarious organs occur due to the autoimmune system. As an onset mechanismof these diseases, there has been considered the enhanced allergicreactions caused by Th2 type cytokines such as interleukin-4 andinterleukin-5 that the Th2 cells among the Th differentiate involved incellular immune responses against antigen (Progress in Medicine 17:19-20, 1997). The elucidation of the induction mechanism and the controlmechanism has important physiological and pharmacological implications,but detailed mechanisms thereof have yet to be clarified. Therapies ofthese diseases in current use include evasion from antigen, the controlof non-specific inflammatory reactions by the oral administration ofantihistamines that antagonize the binding of mediators such ashistamine to receptors and by topical steroid (Igaku No Ayumi (Journalof Clinical and Experimental Medicine) 180: 51-55, 1997).

[0003] For the treatment of allergic diseases, there can be conceivedthe suppression of allergic reactions by shifting the Th2 typecytokine-dominant allergic state to the Th1 type cytokine-dominantstate, and since interferon-γ produced by Th1 cells suppresses theeffect of enhancing IgE production by interleukin-4 produced by Th2cells (Progress in Medicine 17: 19-20, 1997), interferon-γ has been intrial use for the treatment of allergic diseases (J. Am. Acad. Dermatol.32: 684-685, 1991; Allergy 49: 120-128, 1994; Acta Derm. Venereol 73:130-132, 1993), but the effect is small and the results have not beensatisfactory, and thus has not been subjected to clinical uses.

[0004] For the establishment of atopic diseases, the maintenance of theTh2 type-dominant immunological state and the maintenance of ensuinginflammatory reactions are involved. For the improvement of the Th2type-cytokine dominant immunological state, control of cytokines byimmunosupprresive agents has been attempted (Br. J. Dermatol. 143:365-72, 2000; J. Allergy Clin. Immunol. 106 (1 pt2): S58-64, 2000), butthat did not lead to the essential improvement of the immunologicalstate and thereby had a limited effect.

[0005] On the other hand, BCG vaccine is a vaccine that utilizes anattenuated strain of Mycobacterium bovis and is the only live vaccineapproved for Mycobacterium tuberculosis infections. BCG vaccine has apotent adjuvant effect with little side effects, and thus has been givento many people in the world today as a safe vaccine. Now, BCG vaccinehas been reported to have an activity of shifting CD4+ helper T cells toTh1 cells that are responsible for cell-mediated immunity by producinginterferon-γ and interleukin-2 (Cancer Immunol. Immunother. 39: 401-406,1994; ibid. 40: 103-108, 1995).

[0006] As a protein that is omnipresent among Mycobacteria, the antigen85 complex was identified. The protein complex is composed of an antigen85 complex-forming protein 85A with a molecular weight of about 30-32 kd(Infect. Immun. 57:3123-3130, 1989), an antigen 85 complex-formingprotein 85B (J. Bacteriol. 170: 3847-3854, 1988) and an antigen 85complex-forming protein 85C (Infect. Immun. 59: 3205-3212, 1991), whichare major secretory proteins of Mycobacteria. These secretory proteinsexhibit high homology of the gene sequence and the amino acid sequenceand cross reactivity to monoclonal antibodies among the bacteria of thesame genera such as Mycobacterium tuberculosis, Mycobacterium bovis,Mycobacterium kansasii etc. irrespective of the species (Microbiol. Rev.56: 648-661, 1992) and have, as common functions, the activity ofbinding to fibronectin and of mycolyl group transferase in the cell wallsynthesis (Microbiol. Rev. 56: 648-661, 1992; Science 276: 1420-1422,1997; Nat. Struct. Biol. 7: 887-88, 2000). Among these secretoryproteins, the antigen 85 complex-forming protein 85B is widely known asα antigen.

[0007] Currently, furthermore, α antigen has been isolated and purifiedas a tuberculin reactive protein from the culture supernatant ofMycobacterium tuberculosis, and it has been revealed, there is anepitope in the molecule (Am. Rev. Respir. Dis. 130: 647-649, 1984; ibid.132: 173-174, 1985; Microbiological Reviews 56: 648-661, 1992), and ithas been reported that this α antigen has the above-mentioned effect ofshifting to the Th1 cells (Infect. Immun. 60: 2880-2886, 1992). Attemptshave also been made to use and improve Bacillus Calmette-Guerin byrecombinant DNA technology, and then to use as a vaccine to variouspathogens. For example, it has been reported, an expression vector wasconstructed in which a gene encoding the surface antigen of AIDS viruswas integrated into the gene containing the α antigen, and the vectorwas used to transform Bacillus Calmette-Guerin, which transformant isused as a BCG vaccine (WO 96/4009).

[0008] Up until today, however, no attempts have been made to use a geneencoding the α antigen or the α antigen per se for the treatment ofallergic diseases. Furthermore, though the α antigen has been reportedto have the effect of shifting CD4+ helper T cells to Th1 cells, it isnot yet clear whether the α antigen or the α antigen gene is effectivefor the prevention or treatment of allergic diseases such as atopicdermatitis and asthma for which the mechanism of onset has not beenelucidated. In addition, as described above, though the shifting of theTh1 type/Th2 type balance to the Th2 type-dominant state is consideredto be important for the establishment of allergic diseases, no reportshave been made so far that the mere shifting to the Th1 cell side led tothe improvement of skin conditions of atopic dermatitis.

DISCLOSURE OF THE INVENTION

[0009] Thus, it is an object of the present invention to provide novelpharmaceutical use of the Mycobacterium-derived α antigen such as BCGbacteria or analogs thereof or genes encoding them for the prevention ortreatment of allergic diseases.

[0010] The present inventors have found that when an expression vectorcontaining the α antigen-coding gene is applied to Caspase-1 transgenicmice, a mouse model which is at the Th2 type cytokine-dominantimmunological state and which has a persistent atopic dermatitis-likedermatitis, the production of interleukin-4 was inhibited, blood levelsof histamine and IgE were also suppressed, and skin diseases wereimproved, indicating that atopic dermatitis can be healed. The α antigenprotein was also found to heal atopic dermatitis in a similar manner.Furthermore, when an expression vector containing the α antigen-codinggene was applied to a mouse asthma model, it was found, IgE productionwas inhibited and allergic conditions such as eosinophil-infiltrationcan be improved, leading to the healing of asthma. Thus, the presentinventors have revealed that the α antigen gene or the α antigen proteinimproves the Th2 type cytokine-dominant immunological state and cansuppress and/or improve various conditions of allergic diseases, andthus is widely effective for the prevention or treatment of allergicdiseases, and thereby have completed the present invention.

[0011] Thus, the present invention relates to a pharmaceuticalcomposition for the prevention or treatment of allergic diseasescomprising, as an active ingredient, the Mycobacterium-derived αantigen, an analog thereof, a mutant thereof having a function similarthereto, or a gene encoding them.

[0012] Furthermore, the present invention relates to a method ofpreventing or treating allergic diseases, said method comprisingadministering an effective amount of the Mycobacterium-derived αantigen, an analog thereof, a mutant thereof having a function similarthereto, or a gene encoding them to mammals including humans.

[0013] Furthermore, the present invention relates to the use of theMycobacterium-derived α antigen, an analog thereof, a mutant thereofhaving a function similar thereto, or a gene encoding them for theproduction of a pharmaceutical composition for the prevention ortreatment of allergic diseases.

[0014] According to a preferred embodiment, the present invention usesan expression vector encoding the Mycobacterium-derived α antigen or ananalog thereof, or the α antigen protein or an analog protein thereoffor the prevention or treatment of atopic diseases such as atopicdermatitis, asthma, allergic rhinitis, and allergic conjunctivitis. Asused herein, as analogs of the α antigen, there can be mentioned anantigen 85 complex-forming protein 85A, antigen 85 complex-formingprotein 85C and the like.

BRIEF EXPLANATION OF THE DRAWINGS

[0015]FIG. 1 shows the construction of an expression vector, constructedin Example 1, containing a gene encoding the α antigen for use as anactive ingredient of a pharmaceutical composition for the prevention ortreatment of allergic diseases of the present invention.

[0016]FIG. 2 is a drawing showing the effect of an expression vectorcontaining a gene encoding the α antigen on the treatment of atopicdermatitis.

[0017]FIG. 3 is a graph showing serum levels of IgE when an expressionvector containing a gene encoding the α antigen was administered to amouse asthma model.

[0018]FIG. 4 is a graph showing protein concentrations in the alveolarlavage when an expression vector containing a gene encoding the αantigen was administered to a mouse asthma model.

[0019]FIG. 5 is a graph showing eosinophil counts in the alveolar lavagewhen an expression vector containing a gene encoding the α antigen wasadministered to a mouse asthma model.

[0020]FIG. 6 is a graph showing the degree of infiltration ofeosinophils in the lung tissue when an expression vector containing agene encoding the α antigen was administered to a mouse asthma model.

[0021]FIG. 7 is a graph showing the result of histological examinationof the lung when an expression vector containing the gene encoding the αantigen was administered to a mouse asthma model.

BEST MODE FOR CARRYING OUT THE INVENTION

[0022] The subject diseases of the present invention are allergicdiseases, and more specifically allergic diseases caused by the Th2 typecytokine-dominant state. The preferred subject allergic diseases of thepresent invention are specifically atopic diseases, for example atopicdermatitis, asthma, allergic rhinitis and allergic conjunctivitis, andspecifically atopic dermatitis and asthma.

[0023] As used herein, genes encoding the Mycobacterium-derived αantigen or an analog thereof refers to genes capable of expressing the αantigen protein, or α antigen protein analogs such as antigen 85complex-forming protein 85A and antigen 85 complex-forming protein 85C.Specifically, there can be mentioned genes in the form of an expressionvector containing a gene encoding the α antigen or an analog thereof. Asgenes encoding the α antigen, there can be illustrated genes encodingthe α antigen derived from Mycobactera such as Mycobacterium kansasii(Infect. Immun. 58: 550-556, 1990), Mycobacterium avium (Infect. Immun.61: 1173-1179, 1993), Mycobacterium intracellulare (Biochem. Biophys.Res. Commun. 196: 1466-1473, 1993), Mycobacterium leprae (Mol.Microbiol. 6: 153-163, 1992) and the like. Any of these genes can beused in the present invention, and as a gene encoding α antigen derivedfrom Mycobacterium kansasii, there can be mentioned a DNA having thebase sequence from positions 390 to 1244 of SEQ ID NO: 1.

[0024] In addition to this DNA, they may be a mutant DNA that hybridizesto this DNA under a stringent condition, or a mutant DNA comprising aDNA encoding a protein having an amino acid sequence in which one ormore than one (preferably several) amino acid residues have beensubstituted, deleted and/or added to the amino acid sequence of theprotein encoded by this DNA, wherein said mutant encodes a proteinhaving the same function as the Mycobacterium kansasii-derived αantigen. As used herein, as a specific method of obtaining a mutant DNAthat hybridizes to this DNA under a stringent condition, there can bementioned the following method. Thus, a colony hybridization isperformed in the presence of 50% formamide, 4× Denhardt, 5×SSPE (SSPEsolution: EDTA sodium phosphate (SSPE),1× Denhardt: 0.02% Ficoll, 0.02%polyvinyl pyrrolidone, 0.02% bovine serum albumin), 0.2% SDS, 100 μg/mlssDNA, and 12.5 ng of a probe (12.5 ng of a purified cDNA fragmenthaving a base sequence from positions 390 to 1244 of SEQ ID NO: 1labelled with [α=³²P]dCTP (Amersham) using the BcaBest DNA Labeling kit(TaKaRa)) at 45° C. for 14-16 hours, the filter is washed in 1×SSPE anda 0.5% SDS solution at 45° C./30 min, then in 0.1×SSPE and a 0.5% SDSsolution at 55° C./1 hour, and finally 0.1×SSPE and a 0.5% SDS solutionat 65° C./1 hour to eliminate the background completely, which is thenexposed to an X-ray film (Fuji) at −80° C. for 72 hours to determine theposition of and isolate the corresponding colony and thus the mutant DNAcan be obtained. The above same function as the α antigen derived fromMycobacterium kansasii means to have a similar effect of preventing ortreating allergic diseases. These mutants are those for which amino acidsequences encoded thereby usually have a homology of 60% or greater withthe amino acid sequence of the α antigen protein, and preferably ahomology of 75% or greater. In the case of genes encoding the α antigenother than Mycobacterium kansasii, they may be mutants thereof as well.

[0025] As genes encoding the antigen 85 complex-forming protein 85Awhich is an analog of the α antigen, there can be mentioned genesderived from the Mycobacteria similar to various Mycobacteria describedabove for the α antigen gene. More specifically, there can be mentioneda DNA encoding the antigen 85 complex-forming protein 85A derived fromMycobacterium tuberculosis (Infect. Immun. 57: 3123-3130, 1989). Forgenes encoding the antigen 85 complex-forming protein 85C as well, therecan be mentioned a gene derived from various Mycobacteria, and morespecifically, there can be mentioned a DNA encoding the antigen 85complex-forming protein 85C derived from Mycobacterium tuberculosis(Infect. Immun. 59: 3205-3212, 1991). For these DNAs as well, asdescribed antibody, they may be a mutant DNA that hybridizes to this DNAunder a stringent condition, or a mutant DNA comprising a DNA encoding aprotein having an amino acid sequence in which one or more than one(preferably several) amino acid residues have been substituted, deletedand/or added to the amino acid sequence of the protein encoded by thisDNA, wherein said mutant encodes a protein having the same function asthe antigen 85 complex-forming protein 85A or the antigen 85complex-forming protein 85C.

[0026] The above DNAs can be cloned by a methyl such as RT-PCR reactionon mRNA derived from Mycobacteria using as PCR primers appropriate DNAsegments based on the sequence information described in the aboveliterature, sequence information of Genebank, etc. Chemical synthesis isalso possible based on the amino acid sequence information. Furthermore,the above DNA mutants may be easily obtained using, for example,site-directed mutagenesis, PCR method, a common hybridization method, orthe like.

[0027] In accordance with the present invention, the α antigen proteinderived from Mycobacteria or derivatives thereof such as antigen 85complex-forming protein 85A or antigen 85 complex-forming protein 85C ormutant proteins thereof can be used for the prevention or treatment ofallergic diseases. As such α antigens, there can be mentioned proteinsencoded by genes encoding the above-mentioned α antigen. Specifically,there can be mentioned an α antigen which is encoded by the DNA havingthe base sequence derived from Mycobacterium kansasii described in SEQID NO: 1 and which has the amino acid sequence of SEQ ID NO: 2. Inaddition to the α antigen having this amino acid sequence, it may be amutant protein comprising an amino acid sequence in which one or morethan one (preferably several) amino acid residues have been substituted,deleted and/or added to the amino acid sequence of SEQ ID NO: 2, saidprotein having the same function as the α antigen. In the case of the αantigen derived from Mycobacterium other than Mycobacterium kansasii aswell, it may be a mutant protein comprising a protein in which one ormore than one (preferably several) amino acid residues have beensubstituted, deleted and/or added to the amino acid sequence of thoseamino acid sequences, said protein having the same function as the αantigen. For the antigen 85 complex-forming protein 85A or the antigen85 complex-forming protein 85C as well, there can be mentioned theantigen 85 complex-forming protein 85A derived from Mycobacteriumtuberculosis (Infect. Immun. 57: 3123-3130, 1989), the antigen 85complex-forming protein 85C derived from Mycobacterium tuberculosis(Infect. Immun. 59: 3205-3212, 1991) and the like. The analogs of theseα antigen proteins may be mutants similar to those of the α antigenprotein mentioned above.

[0028] These proteins may be produced by a recombinant DNA technologyusing genes encoding them or by chemical synthesis. Alternatively,Mycobacteria such as Mycobacterium kansasii may be cultured in asuitable medium, and from the culture liquid, the proteins may bepurified by a known purification method (Scand. J. Immunol. 43: 202-209,1996; J. Bacteriol. 170: 3847-3854, 1988; Hiroshima J. Med. Sci. 32:1-8, 1983).

[0029] In accordance with the present invention, when genes encoding theMycobacterium-derived α antigen, a derivative thereof, or a mutantthereof are used for the prevention or treatment of allergic diseases,specifically they may be used in the form of an expression vectorcontaining a gene encoding the α antigen, an analog thereof, or a mutantthereof. Expression vectors containing these genes are roughly dividedinto two: cases when non-virus vectors are used, and cases when virusvectors are used.

[0030] Non-virus vectors may be any expression vectors as long as theycan express and secrete genes that encode the α antigen, a derivativethereof, or a mutant thereof, and by way of example, there can bementioned pCAGGS (Gene 108: 193-200, 1991), pBK-CMV, pcDNA3.1, pZeoSV(Invtrigen, Stratagene) etc.

[0031] As virus vectors, representative virus vectors includerecombinant adenovirus, retrovirus and the like. More specifically,there can be mentioned DNA viruses or RNA viruses such as detoxicatedretrovirus, adenovirus, adeno-associated virus, helper virus, vacciniavirus, pox virus, poliovirus, Sindbis virus, Sendai virus, SV40, andhuman immunodeficiency virus (HIV).

[0032] By integrating a gene encoding the α antigen, an analog thereofor a mutant thereof into these vectors, expression vectors can beconstructed.

[0033] These vectors may be generally administered to mammals includinghumans in the form of injections depending on the method of introducinginto the living body. In the case of virus vectors, they may beadministered as they are. Injections may be prepared according to astandard method, and for example after they are dissolved in a suitablesolvent (a buffer such as PBS, physiological saline, sterile wateretc.), they may be filter-sterilized as desired with a filter etc. andthen filled into a sterile container for formulation. Commonly usedcarriers may be added to the injections, as necessary. They may also bein the form of liposome formulations described below.

[0034] In order to use the expression vectors thus obtained for thetreatment of allergic diseases, they can be administered in a standardmethod. Specifically, such methods include, for example, the lipofectionmethod, the phosphate-calcium coprecipitation method, the DEAE-dextranmethod, the electroporation method, direct DNA injection methods usingmicro glass capillaries, and the like. Furthermore, there are methods ofintroducing genes into the tissue. Such methods include, for example,gene introduction with internal type liposomes, gene introduction withelectrostatic type liposomes, HVJ-liposome methods, improvedHVJ-liposome methods (HVJ-AVE liposome methods), receptor-mediated geneintroduction, methods of introducing DNA molecules into the celltogether with carriers (metal particles) by a particle gun, in vivoelectroporation, and the like. There can also be used a method ofdissolving a non-virus vector expression plasmid into physiologicalsaline and using it as it is (the so-called naked-DNA directintroduction), a method of introduction with positively chargedpolymers, and the like.

[0035] When the expression vector is a virus vector, it can be used asit is, or can be administered in the form of an injection as describedabove.

[0036] Expression vectors containing the gene encoding α antigen, ananalog thereof or a mutant thereof are usually administered to the skin,muscles, the abdominal cavity etc. of mammals including humans. Thedosage may vary depending on the type of the expression vector, dosageform, dosage regimen, the subject patient, type of disease etc., and isgenerally, as the expression vector, about 0.005 to about 2 mg,preferably about 0.1 mg to about 1 mg, generally once daily over severalmonths for a total of a few times.

[0037] In accordance with the present invention, whenMycobacterium-derived α antigen protein, an analog protein thereof, or amutant protein thereof is used as it is, it may generally beadministered pareterally, for example intravenously, intramuscularly,intraperitoneally, subcutaneously, topically, etc. When administeredparenterally, it may be given in the form of an injection, localapplication form, etc.

[0038] As injections, there can be mentioned sterile solutions,suspensions, or the like. As topical applications, there can bementioned creams, ointments, lotions, sprays, aerosols, transdermalformulations (common patches, matrices etc.), and the like. Theseformulations may be prepared by conventional methods in combination withpharmaceutically acceptable excipients, additives etc. Aspharmaceutically acceptable excipients and additives, there can bementioned carriers, binders, flagrants, buffers, thickeners, colorants,stabilizers, emulsifying agents, dispersants, suspending agents,preservatives, and the like.

[0039] Specifically, as injections, there can be mentioned solutions,suspensions, and emulsions and the like. For example, the α antigenprotein may be added into a PBS buffer, physiological saline, sterilewater etc., to which albumin may be added as desired, which isfilter-sterilized with a filter etc. and then filled into sterilecontainers for formulation. They may also be lyophilized and dissolvedto prepare injections at the time of administration. Ointments andcreams used as topical applications may be formulated by adding the αantigen protein together with a thickener or a gelling agent to anaqueous or oily base. As the base, there can be mentioned for examplewater, liquid paraffin, plant oils (peanut oil, castor oil, etc.) andthe like. As thickeners, there can be mentioned for example softparaffin, aluminum stearate, cetostearyl alcohol, polyethylene glycol,lanoline, hydrogenated lanoline, beeswax, and the like. Lotions may beformulated in a standard method by adding one or more ofpharmaceutically acceptable stabilizers, suspending agents, emulsifyingagents, dispersants, thickeners, colorants, flagrants etc. to an aqueousor oily base. Sprays, aerosols, patches, matrices, etc. can also beformulated according to standard methods. These local applications maycontain, as desired, preservatives such as methyl hydroxybenzoate,propyl hydroxybenzoate, chloro cresol, and benzalkonium chloride, andbactericidal agents.

[0040] The dosage and the number of administration of the α antigenprotein, a derivative thereof, or a mutant proteins thereof may varydepending on the symptom, age, body weight, dosage regimen, etc., andwhen administered as injections, generally about 1 mg to about 10 mg,preferably about 1 mg to about 5 mg is administered once or severaltimes in divided doses. When topically administered, generally about 10μg of the dose may be administered over several days.

[0041] The present invention will now be specifically explained withreference to the following examples, but these examples should not beconstrued to limit the present invention.

EXAMPLE 1 Effect of an Expression Vector Containing an αAntigen-Encoding Gene on Atopic Dermatitis

[0042] (1) Construction of Expression Vector

[0043] By inserting the α antigen gene (α-K) of Mycobacterium kansasiicomprising the base sequence from positions 390-1244 set forth in SEQ IDNO: 1 into the KpnI-ApaI site of pcDNA3.1 (Invitrogen CA), and thenintegrating it downstream of the CMV promoter and the TPA signalpeptide, an expression vector pcDNA-α-K having the construction shown inFIG. 1 was constructed as an active ingredient of a pharmaceuticalcomposition for the treatment of allergic diseases of the presentinvention (Infect. Immun. 58: 550-556, 1996).

[0044] (2) Administration of an Expression Vector to a Mouse Model ofAtopic Dermatitis

[0045] i) Method

[0046] Transgenic mice (CTg) that express skin-specific Caspase-1 andthat are in the Th2-dominant immunological state were used (J. Immunol.165: 997-1003, 2000; Nat. Immunol. 1: 132-137, 2000; Proc. Nah. Acod.Sci. USA 99;11340-11345, 2002). These mice developed atopic dermatitisfrom week 8 after birth.

[0047] To CTg mice at week 4, 10 and 12 (4, 10 and 12 week old) afterbirth, 100 μg of the expression vector constructed in the above (1)dissolved in PBS was intraperitoneally administered. Then, for the 4, 10and 12 week old CTg mice, at week 8 after the administration, serumlevels of histamine and IgE as well as the expression level ofinterleukin-4 mRNA in the skin were investigated. Levels of histamineand IgE were measured by RIA, and interleukin-4 mRNA by RT-PCR.

[0048] ii) Result

[0049] The result obtained is shown in Table 1. As can,be seen fromTable 1, in the CTg mouse group that received the expression vector, noincreases in blood levels of IgE or histamine were observed, and were inthe normal range. Also, interleukin-4 mRNA in the skin becameundetectable. During the observation period, no development ofdermatitis or no scratch behavior were seen.

[0050] In contrast, no treatment CTg mice developed dermatitis at week 8and exhibited scratch behavior. Also, blood levels of IgE and histaminewere elevated. TABLE 1 Effect on atopic dermatitis IgE level (μg/ml)Histamine (nM/1) Littermate Skin IL-4 mRNA Time of Before 8 weeks afterno treatment 8 weeks after Before 8 weeks after administrationadministration administration mice administration administrationadministration  4 week old 158 15 16 0 ++ — 10 week old 230 47 19 0 ++ —12 week old 200 170 35 0 ++ —

[0051] In FIG. 2, photographs of various CTg mice are shown. Aphotograph in top left shows CTg mice at the onset of atopic dermatitis,and one in top right shows 12 week old littermate CTg mice that receivedno treatment. A photograph in bottom left shows a result in which alittermate CTg mice was cured by the intramuscular injection ofprednisolone for 7 days. A photograph in bottom right shows the effectof treatment on week 8 after the intraperitoneal administration of theexpression vector constructed in the above (1) to 4 week old CTg mice.After the administration of the expression vector, no development ofdiseases was seen during the observation period (one year). Thesephotographs clearly indicate that the administration of the expressionvector containing the α antigen-encoding gene can very effectivelyprevent the onset of or treat atopic dermatitis.

EXAMPLE 2 Effect of the α Antigen Protein on Atopic Dermatitis

[0052] i) Method

[0053] Mycobacterium kansasii was cultured in the Sauton medium for 3weeks, and the culture supernatant was precipitated with 80% ammoniumsulfate to prepare a protein fraction. The protein fraction wasdeveloped and separated by two dimensional electrophoresis on the gel,and from the corresponding spot on the gel, α antigen protein wasextracted and purified (Scand. J. Immunol. 43: 202-209, 1996; J.Bacteriol. 170: 3847-3854, 1988; Hiroshima J. Med. Sci. 32: 1-8, 1983),and dissolved in PBS at a concentration of 1 mg/ml. One μl each of thisα antigen protein solution or the control PBS solution was applied onthe head of 4 week old CTg mice (N=3 for each) once daily for one week,and then the condition of hair was visually inspected to asses theeffect.

[0054] ii) Result

[0055] When the effect was judged by the amount of remaining hair as +(hair growth is seen) or − (no hair growth is seen), hair on the headwas scratched away (due to itching) and the festered skin was exposed(−) in the control to which the PBS solution was only applied, whereasin the α antigen protein-application group hair on the head remained asit was (+ to ++).

EXAMPLE 3 Effect of Expression Vector Containing the α Antigen-EncodingGene on Asthma

[0056] 1) Method

[0057] BALB/c mice (N=6 for each group) were immunized withintraperitoneally administration of 10 μg of ovalbumin and 1 mg alum onday 0 and day 14 after the start of the experiment. Fot five days fromday 21, the animals were allowed to inhale aerosol of 5% ovalbumin tocreate an asthma model. The expression vector (100 μg) constructed inExample 1-(I) and the heated BCG dead organism (100 μg) wereintraperitoneally administered in equal amounts, respectively, on day 0and day 14. On day 25 after the start of the experiment, serum levels ofIgE, protein concentration in the alveolar lavage, and eosinophil countswere determined for judgement of effect, and furthermore eosinophilinfiltration in the lung tissue was examined by an eosinophil staining(Luna stain). Also the lung tissue was histologically examined.

[0058] ii) Result

[0059] Serum levels of IgE, protein concentration in the alveolarlavage, eosinophil counts, eosinophil infiltration, and the histologicalimage of the lung are shown in FIG. 3 to FIG. 7, respectively. As can beseen from the result in FIG. 3, serum levels of IgE significantlydecreased in the α antigen gene-containing expression vectoradministration group compared to the no treatment group. As can be seenfrom the result in FIG. 4, protein concentration in the alveolar lavagewhich is an index of inflammatory reaction was apparently low in theexpression vector administration group compared to the no treatmentgroup and the BCG administration group. As can be seen from the resultin FIG. 5, in the expression vector administration group, the number ofeosinophils that infiltrate the lung lavage was apparently repressed andthe effect was more pronounced than the BCG administration group. As canbe seen from the result in FIG. 6, infiltration of a large number ofeosinophils was seen in the no treatment group, but not in theexpression vector administration group. As can be seen from the resultin FIG. 7, apparent allergic inflammation was noted in the no treatmentgroup whereas the expression vector administration group exhibitedlittle difference from the normal mice.

[0060] From the foregoing, in the α antigen gene-containing expressionvector administration group, a marked effect of suppressing asthmasymptoms was noted close to the no treatment normal mice, and the effectwas considered to be greater than the administration of an equal amountof BCG.

INDUSTRIAL APPLICABILITY

[0061] As described above, when the α antigen, an analog thereof, or anexpression vector containing the gene encoding those analogs isadministered to atopic dermatitis, histamine release is inhibited, andfurthermore the production of IgE and interleukin-4 is suppressed,improving skin diseases and thereby exhibiting a highly effective effectfor the treatment of atopic dermatitis. Furthermore, the α antigen, ananalog protein thereof or an analog protein thereof also exhibits highlyeffective effect for the treatment of atopic dermatitis. The α antigen,an analog protein thereof or an analog protein thereof is considered toexhibit the effect of improving the Th2 type cytokine-dominantimmunological state, and therefore the α antigen, an analog thereof orthe gene encoding an analog thereof, and the α antigen protein, ananalog protein thereof or a mutant protein thereof is very effective forthe prevention or treatment of atopic diseases such as atopicdermatitis, asthma, allergic rhinitis and allergic conjunctivitis, andmore broadly allergic diseases.

1. A pharmaceutical composition for the prevention or treatment ofallergic diseases comprising as an active ingredientMycobacterium-derived α antigen, an analog thereof, a mutant having afunction similar thereto, or a gene encoding one of them.
 2. Thepharmaceutical composition for the prevention or treatment of allergicdiseases of claim 1 wherein the gene is in the form of an expressionvector.
 3. The pharmaceutical composition for the prevention ortreatment of allergic diseases of claim 1 or 2 wherein the α antigen isMycobacterium kansasii-derived α antigen.
 4. The pharmaceuticalcomposition for the prevention or treatment of allergic diseases ofclaim 1 or 2 wherein the analog of the α antigen is the antigen 85complex-forming protein 85A or the antigen 85 complex-forming protein85C.
 5. The pharmaceutical composition for the prevention or treatmentof allergic diseases of any one of claims 1 to 4 wherein allergicdiseases are those allergic diseases that are caused by the Th2 typecytokine-dominant state.
 6. The pharmaceutical composition for theprevention or treatment of allergic diseases of any one of claims 1 to 5wherein allergic diseases are atopic diseases.
 7. The pharmaceuticalcomposition for the prevention or treatment of allergic diseases of anyone of claims 1 to 6 wherein allergic diseases are atopic dermatitis,asthma, allergic rhinitis, or allergic conjunctivitis.
 8. Thepharmaceutical composition for the prevention or treatment of allergicdiseases of any one of claims 1 to 7 wherein allergic diseases areatopic dermatitis or asthma.
 9. A method of preventing or treatingallergic diseases comprising administering an effective amount ofMycobacterium-derived α antigen, an analog thereof, a mutant having afunction similar thereto, or a gene encoding one of them to mammalsincluding humans.
 10. The method of preventing or treating allergicdiseases of claim 9 wherein the gene is in the form of an expressionvector.
 11. The method of prevention or treatment of claim 9 wherein theα antigen is Mycobacterium kansasii-derived α antigen.
 12. The method ofprevention or treatment of any of claims 9 to 11 wherein the analog of αantigen is the antigen 85 complex-forming protein 85A or the antigen 85complex-forming protein 85C.
 13. The method of prevention or treatmentof any of claims 9 to 12 wherein allergic diseases are atopicdermatitis, asthma, allergic rhinitis, or allergic conjunctivitis. 14.The use of Mycobacterium-derived α antigen, an analog thereof, a mutanthaving a function similar thereto, or the gene encoding one of them forthe production of a pharmaceutical composition for the prevention ortreatment of allergic diseases.
 15. The use of claim 14 wherein the geneis in the form of an expression vector.
 16. The use of claim 14 or 15wherein the α antigen is Mycobacterium kansasii-derived α antigen. 17.The use of a any of claims 14 to 16 wherein the analog of α antigen isthe antigen 85 complex-forming protein 85A or the antigen 85complex-forming protein 85C.
 18. The use of any of claims 14 to 17wherein allergic diseases are atopic dermatitis, asthma, allergicrhinitis, or allergic conjunctivitis.